Porcine high molecular weight kininogen: its purification and properties as a thiol proteinase inhibitor as compared to human high molecular weight kininogen

1. High mol. wt kininogen (HMW kininogen) was purified to a homogeneous state from porcine plasma. 2. The protein exhibited a strong inhibitory activity for thiol proteinases such as ficin, papain and calpain I, whereas it did not inhibit serine proteinases, trypsin and chymotrypsin. 3. The mol. wt, isoelectric point, amino acid and carbohydrate compositions, stabilities to temperature and pH, kinetic constants, and immunological properties of the porcine HMW kininogen were determined and compared with those of human HMW kininogen.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Oxidative degradation of thymine with O2 promoted by L-ascorbic acid and Cu(II) ion

Oxidation of thymine with O2 was promoted by copper(I) ion generated from reaction of L-ascorbic acid (AA) with copper (II) ion. The main oxidation products were thymine glycol (TG) and N-formyl-N'-pyruvylurea (FPU). At higher concentration of O2, formation of FPU was favored, while TG was dominant at higher Cu(II) ion and lower O2 concentrations. Reaction mechanism involving hydroxy thyminyl radical was proposed.     

Reduced Visual Cortex Gray Matter Volume and Thickness in Young Adults Who Witnessed Domestic Violence during Childhood

Exposure to interparental violence is associated with negative outcomes, such as depression, post-traumatic stress disorder and reduced cognitive abilities. However, little is known about the potential effects of witnessing domestic violence during childhood on gray matter volume (GMV) or cortical thickness. High-resolution 3.0 T volumetric scans (Siemens Trio Scanner) were obtained on 52 subjects (18–25 years) including 22 (6 males/16 females) with a history of visually witnessing episodes of domestic violence, and 30 (8 males/22 females) unexposed control subjects, with neither a current nor past DSM-IV Axis I or II disorder. Potential confounding effects of age, gender, level of parental verbal aggression, parental education, financial stress, full scale IQ, and total GMV, or average thickness were modeled using voxel based morphometry and FreeSurfer. Witnessing domestic violence subjects had a 6.1% GMV reduction in the right lingual gyrus (BA18) (P = 0.029, False Discovery Rate corrected peak level). Thickness in this region was also reduced, as was thickness in V2 bilaterally and left occipital pole. Theses regions were maximally sensitive to exposure to witnessing domestic violence between 11–13 years of age. Regional reductions in GMV and thickness were observed in both susceptible and resilient witnessing domestic violence subjects. Results in subjects witnessing domestic violence were similar to previously reported results in subjects with childhood sexual abuse, as the primary region affected was visual cortex. Brain regions that process and convey the adverse sensory input of the abuse may be specifically modified by this experience, particularly in subjects exposed to a single type of maltreatment. Exposure to multiple types of maltreatment is more commonly associated with morphological alterations in corticolimbic regions. These findings fit with preclinical studies showing that visual cortex is a highly plastic structure.     

Solid-state modifications of poly(γ-ethyl-L-glutamate)

Several modification of the arrangements of α-helical molecules were found in the solid films of poly (γ-ethyl-L -glutamate), depending on the casting solvent and the temperature. The helical conformation is somewhat looser than the normal 18-residue, 5-turn α-helix. Using x-ray diffraction, the types of molecular arrangements were classified into tetragonal, pseudohexagonal, and hexagonal ones. Tetragonal packing was observed in the filmm (form T) prepared by casting the solution in trifluorethanol or dichlorethane. The sample obtained from chloroform solution is a well-ordered, pseudohexagonal modification (form I). Forms I and T change into a poorly crystalline form III by annealing at temperatures above 130° C. It is particularly noteworthy that the less-ordered form III exhibits a thermoreversible transition around 110°C into a well-ordered form H with the hexagonal molecular packing.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Stability of 3' dangling ends on the core helix of AUGCAU at various Na+ concentrations.

Stability increments of 3' dangling ends on the core helices AUGCAU at various Na+ concentrations are reported. The results show that all 3' dangling ends except 3'U dangling at low Na+ concentrations can stabilize the helix and this stabilization is very sequence dependent.